Gao H Q, Boyer P L, Sarafianos S G, Arnold E, Hughes S H
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA.
J Mol Biol. 2000 Jul 7;300(2):403-18. doi: 10.1006/jmbi.2000.3823.
Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.
使用阻断病毒复制的药物治疗HIV感染会筛选出该病毒的耐药毒株。特定的抑制剂会筛选出具有特征性的耐药突变。就核苷类似物3TC和FTC而言，耐药病毒是在逆转录酶（RT）的氨基酸残基184处发生突变而被筛选出来的。最初的变化通常是变为M184I；这种病毒会迅速被携带M184V突变的变体所取代。3TC和FTC被细胞摄取并转化为3TCTP和FTCTP。这些核苷类似物的三磷酸形式被HIV-1 RT掺入DNA并充当链终止剂。M184I和M184V这两种突变在体内都具有很高的耐药性；携带M184V和M184I的纯化HIV-1 RT在体外聚合酶测定中也显示出对3TCTP和FTCTP的耐药性。氨基酸M184是HIV-1 RT的dNTP结合位点的一部分。结构研究表明，携带M184V或M184I突变的HIV-1 RT的耐药机制涉及空间位阻，这可能会完全阻断3TCTP和FTCTP的结合，或者允许这些核苷三磷酸分子结合，但只是以一种会阻止掺入的构象。现有的动力学数据并不明确：一组报告称突变的主要作用是在3TCTP结合水平；另一组则认为是在掺入水平。我们通过监测HIV-1 RT在结合dNTP时发生构象变化能力的测定方法来解决这个问题。这些研究表明，野生型RT和耐药变体都能在聚合酶活性位点结合3TCTP；然而，与M184V和M184I的结合稍弱且对盐敏感。我们提出，耐药变体以一种对盐敏感且无催化活性的紧张构象结合3TCTP。
